# Design of solubly expressed miniaturized SMART MHCs

## TL;DR

Scientists designed a new type of MHC protein that can be produced in bacteria and retains the ability to bind T-cells and peptides, helping study immune responses.

## Contribution

The novel SMART MHCs replace β2m and α3 domain, enabling soluble expression and maintaining functional binding properties.

## Key findings

- SMART MHCs retain peptide- and TCR-binding specificity.
- Peptide-bound SMART MHC structures resemble native MHCs.
- SMART MHCs can be produced in E. coli in soluble form.

## Abstract

The precise recognition of specific peptide-MHC (pMHC) complexes by T-cell receptors (TCRs) plays a key role in infectious disease, cancer and autoimmunity. A critical step in many immunobiological studies is the identification of T-cells expressing TCRs specific to a given pMHC antigen. However, the intrinsic instability of empty class-I MHCs limits their soluble expression in Escherichia coli and makes it very difficult to characterize even a small fraction of possible pMHC/TCR interactions. To overcome this limitation, we designed small proteins which buttress the peptide binding groove of class I MHCs, replacing β2-microglobulin (β2m) and the heavy chain α3 domain, and enable soluble and partially soluble expression in E. coli of H-2Db and A*02:01, respectively. We demonstrate that these soluble, monomeric, antigen-receptive, truncated (SMART) MHCs retain both peptide- and TCR-binding specificity, and that peptide-bound structures of both allomorphs are similar to their full-length, native counterparts. With extension to the majority of HLA alleles, SMART MHCs should be broadly useful for probing the T-cell repertoire in approaches ranging from yeast display to T-cell staining.